Mtb Burden Research Articles

SIV and Mycobacterium tuberculosis synergy within the granuloma accelerates the reactivation pattern of latent tuberculosis.

Human immunodeficiency virus infection is the most common risk factor for severe forms of tuberculosis (TB), regardless of CD4 T cell count. Using a well-characterized cynomolgus macaque model of human TB, we compared radiographic, immunologic and microbiologic characteristics of early (subclinical) reactivation of latent M. tuberculosis (Mtb) infection among animals subsequently infected with simian immunodeficiency virus (SIV) or who underwent anti-CD4 depletion by a depletion antibody. CD4 depleted animals had significantly fewer CD4 T cells within granulomas compared to Mtb/SIV co-infected and Mtb-only control animals. After 2 months of treatment, subclinical reactivation occurred at similar rates among CD4 depleted (5 of 7 animals) and SIV infected animals (4 of 8 animals). However, SIV-induced reactivation was associated with more dissemination of lung granulomas that were permissive to Mtb growth resulting in greater bacterial burden within granulomas compared to CD4 depleted reactivators. Granulomas from Mtb/SIV animals displayed a more robust T cell activation profile (IFN-α, IFN-γ, TNF, IL-17, IL-2, IL-10, IL-4 and granzyme B) compared to CD4 depleted animals and controls though these effectors did not protect against reactivation or dissemination, but instead may be related to increased viral and/or Mtb antigens. SIV replication within the granuloma was associated with reactivation, greater overall Mtb growth and reduced Mtb killing resulting in greater overall Mtb burden. These data support that SIV disrupts protective immune responses against latent Mtb infection beyond the loss of CD4 T cells, and that synergy between SIV and Mtb occurs within granulomas.

Small Animal Model of Post-chemotherapy Tuberculosis Relapse in the Setting of HIV Co-infection.

Tuberculosis relapse following drug treatment of active disease is an important global public health problem due to the poorer clinical outcomes and increased risk of drug resistance development. Concurrent infection with HIV, including in those receiving anti-retroviral therapy (ART), is an important risk factor for relapse and expansion of drug resistant Mycobacterium tuberculosis (Mtb) isolates. A greater understanding of the HIV-associated factors driving TB relapse is important for development of interventions that support immune containment and complement drug therapy. We employed the humanized mouse to develop a new model of post-chemotherapy TB relapse in the setting of HIV infection. Paucibacillary TB infection was observed following treatment with Rifampin and Isoniazid and subsequent infection with HIV-1 was associated with increased Mtb burden in the post-drug phase. Organized granulomas were observed during development of acute TB and appeared to resolve following TB drug therapy. At relapse, granulomatous pathology in the lung was infrequent and mycobacteria were most often observed in the interstitium and at sites of diffuse inflammation. Compared to animals with HIV mono-infection, higher viral replication was observed in the lung and liver, but not in the periphery, of animals with post-drug TB relapse. The results demonstrate a potential role for the humanized mouse as an experimental model of TB relapse in the setting of HIV. Long term, the model could facilitate discovery of disease mechanisms and development of clinical interventions.

Curdlan Limits Mycobacterium tuberculosis Survival Through STAT-1 Regulated Nitric Oxide Production

Host-directed therapies have emerged as an innovative and promising approach in tuberculosis (TB) treatment due to the observed limitations of current TB regimen such as lengthy duration and emergence of drug resistance. Thus, we explored the role of curdlan (beta glucan polysaccharide) as a novel strategy to activate macrophages against Mycobacterium tuberculosis (Mtb). The aim of the study was to investigate the role of curdlan in restricting the Mtb growth both in vitro and in vivo. Further, the immunomodulatory potential of curdlan against Mtb and the underlying mechanism is largely unknown. We found that curdlan treatment enhanced the antigen presentation, pro-inflammatory cytokines, Mtb uptake and killing activity of macrophages. In vivo studies showed that curdlan therapy significantly reduced the Mtb burden in lung and spleen of mice. Administration of curdlan triggered the protective Th1 and Th17 immunity while boosting the central and effector memory response in Mtb infected mice. Curdlan mediated anti-Mtb activity is through signal transducer and activator of transcription-1 (STAT-1), which regulates nitric oxide (NO) production through inducible NO synthase (iNOS) induction; along with this activation of nuclear factor kappa B (NF-κB) was also evident in Mtb infected macrophages. Thus, we demonstrate that curdlan exerts effective anti-tuberculous activity anti-tuberculous activity. It can be used as a potential host-directed therapy against Mtb.

PO 8179 MEAN LEVELS OF ENDOPLASMIC RETICULUM STRESS CHAPERONE PROTEIN – BINDING IMMUNOGLOBULIN PROTEIN (BIP) DECREASES FOLLOWING SUCCESSFUL TB TREATMENT

BackgroundMycobacterium tuberculosis (Mtb) infection is one of the leading causes of mortality worldwide. Even though treatment is readily available the emergence of drug resistance amongst Mtb strains highlights the need for new advances in the TB field such as host-directed therapies (HDT). Recent studies have highlighted the importance of BiP in cells, which can become a target in many diagnostic settings as it has been implicated in conditions including arthritis, cancer, bacterial infection and autoimmune diseases. In our studies, we are aiming to identify expression differences of BiP in different Mtb infection stages to help us understand the change of function in immune cells in relation to infection stress.MethodBiP secretion levels were assessed in plasma samples using ELISA technique. This included participants at TB diagnosis (TBDx), TB treatment group (Week 1, Month 2 and Month 6) and healthy (unexposed) participants. BiP concentration results were analysed using GraphPad Prism 7.ResultsSecretion of BiP was comparable between newly diagnosed untreated TB cases and healthy unexposed controls, with levels obtained in healthy group (42.64 µg/ml) and in TBDx (40.88 µg/ml). Highest levels of plasma BiP during treated TB was observed by Week 1 (mean 68.57 µg/ml) and declined by Month 2 with 60.92 µg/ml and Month 6 with 51.40 µg/ml.ConclusionDetection of BiP in plasma samples indicated metabolic change in immune cells due to stress posed onto cells by Mtb burden. This is due to the amount of protein product required by the immune system to mitigate the spread of the pathogen. Even though not significant, we observed a decrease in the mean levels of BiP over the course of TB treatment which correlates with a reduction in the accumulation of unfolded polypeptides in the endoplasmatic reticulum. This observation requires further testing in larger prospective studies.

Design and synthesis of novel pyrimidine derivatives as potent antitubercular agents

The emergence of various drug-resistant Mycobacterium tuberculosis (Mtb) strains has necessitated the exploration of new drugs that lack cross-resistance with existing therapeutics. By screening the MedChemExpress bioactive compound library, ceritinib was identified as a compound with activity against Mtb H37Ra. Ceritinib had a MIC value of 9.0 μM in vitro and demonstrated in vivo efficacy in a BALB/c mouse model infected with autoluminescent H37Ra. Then, 32 novel ceritinib derivatives were synthesized, and their antimycobacterial activities were evaluated in vitro. The antimycobacterial activities of the synthesized compounds were drastically affected by substitutions at position 4 of the pyrimidine nucleus and were enhanced by the presence of 2-isopropoxy-5-methyl-4-(piperidin-4-yl)aniline at position 2 of the pyrimidine nucleus. The in vivo antitubercular activities of the three most potent compounds were evaluated. 5-Chloro-N2-(2-isopropoxy-5-methyl-4-(piperidin-4-yl) phenyl)-N4-(naph thalen-1-yl) pyrimidine-2,4-diamine (16j) remarkably reduced the Mtb burden of mice. This result suggested the potential of 16j as a novel drug with superior antitubercular activities. The results of experiments on the combination of sulfamethoxazole with 16j and in silico modeling suggest that dihydrofolate reductase is the potential molecular target of 16j.

Deletion of BCG Hip1 protease enhances dendritic cell and CD4 T cell responses.

Dendritic cells (DCs) play a key role in the generation of CD4 T cell responses to pathogens. Mycobacterium tuberculosis (Mtb) harbors immune evasion mechanisms that impair DC responses and prevent optimal CD4 T cell immunity. The vaccine strain Mycobacterium bovis Bacille Calmette-Guérin (BCG) shares many of the immune evasion proteins utilized by Mtb, but the role of these proteins in DC and T cell responses elicited by BCG is poorly understood. We previously reported that the Mtb serine protease, Hip1, promotes sub-optimal DC responses during infection. Here, we tested the hypothesis that BCG Hip1 modulates DC functions and prevents optimal antigen-specific CD4 T cell responses that limit the immunogenicity of BCG. We generated a strain of BCG lacking hip1 (BCGΔhip1) and show that it has superior capacity to induce DC maturation and cytokine production compared with the parental BCG. Furthermore, BCGΔhip1-infected DCs were more effective at driving the production of IFN-γ and IL-17 from antigen-specific CD4 T cells in vitro. Mucosal transfer of BCGΔhip1-infected DCs into mouse lungs induced robust CD4 T cell activation in vivo and generated antigen-specific polyfunctional CD4 T cell responses in the lungs. Importantly, BCGΔhip1-infected DCs enhanced control of pulmonary bacterial burden following Mtb aerosol challenge compared with the transfer of BCG-infected DCs. These results reveal that BCG employs Hip1 to impair DC activation, leading to attenuated lung CD4 T cell responses with limited capacity to control Mtb burden after challenge.

Monitoring the response to TB treatment using T cells biomarkers in TB/HIV patients undergoing ART

Abstract TB is the leading cause of death in HIV-infected persons. Anti-TB treatment in TB/HIV co-infected persons is therefore a public health priority. In patients undergoing anti-TB treatment and ART, ART initiation is often deferred by 2–8 weeks at the physician’s discretion in patients presenting CD4 counts >350 cells/ml to sufficiently reduce the Mtb burden in order to minimize potential drug interactions and complications related to the immune reconstitution inflammatory syndrome (IRIS). However, we currently lack reliable “real time” diagnostic methods to assess Mtb clearance during TB treatment. Isolation of Mtb from sputum can take 4–6 weeks and the rate of sputum smear-negative/active TB cases is upto 66% higher in HIV(+) than in HIV (−) patients. PCR-based technology while sensitive, does not differentiate between live and dead Mtb. We have recently identified specific host immune activation markers (CD38 and HLA-DR) on Mtb-specific CD4+ T cells that correlate with Mtb clearance during and after TB treatment of HIV (−) pulmonary active TB using flow cytometry. We have extended these studies to assess the performance of these biomarkers in diagnosing ATB in HIV(+) patients. Further, we are exploring the value of these biomarker profiles on T cells from TB/HIV co-infected patients undergoing ART and TB treatment, to evaluate their potential to serve as clinical correlates of Mtb clearance in ART treated TB/HIV+ patients.

Species dependent impact of helminth-derived antigens on human macrophages infected with Mycobacterium tuberculosis: Direct effect on the innate anti-mycobacterial response.

BackgroundIn countries with a high prevalence of tuberculosis there is high coincident of helminth infections that might worsen disease outcome. While Mycobacterium tuberculosis (Mtb) gives rise to a pro-inflammatory Th1 response, a Th2 response is typical of helminth infections. A strong Th2 response has been associated with decreased protection against tuberculosis.Principal findingsWe investigated the direct effect of helminth-derived antigens on human macrophages, hypothesizing that helminths would render macrophages less capable of controlling Mtb. Measuring cytokine output, macrophage surface markers with flow cytometry, and assessing bacterial replication and phagosomal maturation revealed that antigens from different species of helminth directly affect macrophage responses to Mtb. Antigens from the tapeworm Hymenolepis diminuta and the nematode Trichuris muris caused an anti-inflammatory response with M2-type polarization, reduced macrophage phagosome maturation and ability to activate T cells, along with increased Mtb burden, especially in T. muris exposed cells which also induced the highest IL-10 production upon co-infection. However, antigens from the trematode Schistosoma mansoni had the opposite effect causing a decrease in IL-10 production, M1-type polarization and increased control of Mtb.ConclusionWe conclude that, independent of any adaptive immune response, infection with helminth parasites, in a species-specific manner can influence the outcome of tuberculosis by either enhancing or diminishing the bactericidal function of macrophages.

Loperamide Restricts Intracellular Growth of Mycobacterium tuberculosis in Lung Macrophages.

New approaches for improving tuberculosis (TB) control using adjunct host-directed cellular and repurposed drug therapies are needed. Autophagy plays a crucial role in the response to TB, and a variety of autophagy-inducing drugs that are currently available for various medical conditions may serve as an adjunct treatment in pulmonary TB. Here, we evaluated the potential of loperamide, carbamazepine, valproic acid, verapamil, and rapamycin to enhance the antimicrobial immune response to Mycobacterium tuberculosis (Mtb). Human monocyte-derived macrophages (MDMs) and murine alveolar cells (MACs) were infected with Mtb and treated with loperamide, carbamazepine, valproic acid, verapamil, and rapamycin in vitro. Balb/c mice were intraperitoneally administered loperamide, valproic acid, and verapamil, and MACs were infected in vitro with Mtb. The induction of autophagy, the containment of Mtb within autophagosomes and the intracellular Mtb burden were determined. Autophagy was induced by all of the drugs in human and mouse macrophages, and loperamide significantly increased the colocalization of microtubule-associated protein 1 light chain 3 with Mtb in MDMs. Carbamazepine, loperamide, and valproic acid induced microtubule-associated protein 1 light chain 3 and autophagy related 16- like protein 1 gene expression in MDMs and in MACs. Loperamide also induced a reduction in TNF-α production. Loperamide and verapamil induced autophagy, which was associated with a significant reduction in the intracellular growth of Mtb in MACs and alveolar macrophages. The intraperitoneal administration of loperamide and valproic acid induced autophagy in freshly isolated MACs. The antimycobacterial activity in MACs was higher after loperamide treatment and was associated with the degradation of p62. In conclusion, loperamide shows potential as an adjunctive therapy for the treatment of TB.

Predictive value of serum bradykinin and desArg9-bradykinin levels for chemotherapeutic responses in active tuberculosis patients: A retrospective case series

BackgroundThere is an urgent need for methods that can rapidly and accurately assess therapeutic responses in patients with active tuberculosis (TB) in order to predict treatment outcomes. Exposure to bacterial pathogens can rapidly activate the plasma contact system, triggering the release of bradykinin (BK) and its metabolite desArg9-bradykinin (DABK) to induce inflammation and innate immune responses. We hypothesized that serum BK and DABK levels might act as sensitive immune response signatures for changes in Mycobacterium tuberculosis (Mtb) burden, and therefore examined how serum levels of these markers corresponded with anti-TB therapy in a small cohort of active TB cases. MethodsNanotrap Mass-Spectrometry (MS) was used to analyze serial blood specimens from 13 HIV-negative adults with microbiologically confirmed active TB who were treated with first-line anti-TB chemotherapy. MS signal for BK (m/z 1060.5) and DABK (m/z 904.5) serum peptides were evaluated at multiple time-points (before, during, and after treatment) to evaluate how BK and DABK levels corresponded with disease status. ResultsSerum BK levels declined from pretreatment baseline levels during the early stage anti-TB therapy (induction phase) and tended to remain below baseline levels during extended treatment (consolidation phase) and after therapy completion. BK levels were consistent with induction phase sputum culture conversions indicative of decreased Mtb burden reflecting good treatment responses. Serum DABK levels tended to increase during the induction phase and decrease at consolidation and post-therapy time points, which may indicate a shift from active disease to chronic inflammation to a disease free state. Elevated BK and DABK levels after treatment completion in one patient may be related to the subsequent recurrent TB disease. ConclusionsOur pilot data suggests that changes in the circulating BK and DABK levels in adult TB patients can be used as potential surrogate markers of the host response both early and late in anti-TB treatment for both pulmonary and extrapulmonary TB patients. We will further exploit these host-response signatures in the future as biomarkers in combination with other clinical and microbiologic tools which may improve treatment efficacy and facilitate the development of host-directed therapy.

Estimating total numbers of Mycobacterium tuberculosis in lymph nodes of cynomolgus and rhesus macaques

Abstract Tuberculosis (TB) is predominantly thought of as a lung disease. However, in classic pathology studies, a thoracic lymph node is often infected in addition to the lungs. Furthermore, lymphadenitis is common in children infected with Mycobacterium tuberculosis (Mtb). In our non-human primate model, at least one lymph node is infected in nearly all macaques. However, rhesus macaques have substantially more lymph node involvement than cynomolgus macaques, and this often leads to clinical deterioration. To better understand the role of lymph nodes in TB, as well as the differences between these species, we quantified the total number of Mtb genomes (live and dead bacteria) in the lymph nodes at 4 and 12 weeks post-infection (p.i.). Mtb burden in the lymph nodes was not significantly different between cynomolgus and rhesus macaques, as well as between 4wk and 12wk p.i.. We then compared the number of Mtb genomes with the number of viable bacteria to determine the degree of bacterial killing in these tissues. Overall, bacterial killing in lymph nodes was very low, compared to lung granulomas. However, the 12wk p.i. rhesus macaques had significantly less Mtb killing in their lymph nodes than the 4wk p.i. rhesus and 12wk p.i. cynomolgus macaques, which could in part explain the greater lymph node involvement in rhesus macaques. The level of bacterial killing between intact and effaced (necrotic) lymph nodes was similar; however, there was a trend of increasing Mtb burden as the effacement increases. Some lymph nodes without culturable bacteria were found to have Mtb genomes present, suggesting lymphatic spread. Our preliminary data suggest that lymph nodes may play a role as a bacterial reservoir and as a means of bacterial dissemination and reactivation.

Autophagy inducing drugs contribute to control Mycobacterium tuberculosis infection

Event Abstract Back to Event Autophagy inducing drugs contribute to control Mycobacterium tuberculosis infection Esmeralda Juárez1*, Claudia Carranza1, Guadalupe Sánchez1, Mitzi González1, Jaime Chávez2, Martha Torres1 and Eduardo Sada1 1 National Institute of Respiratory Diseases, Research in Microbiology, Mexico 2 National Institute of Respiratory Diseases, Research in Bronchial Hyperreactivity, Mexico Tuberculosis is a major cause of morbidity and mortality in the world today. M. tuberculosis (Mtb) escapes from macrophages bactericidal responses. Autophagy is a complex process that ultimately generates a degradative vesicle called autophagosome that has been demonstrated to participate in the control of Mtb infection. Different drugs used for a variety of medical conditions can induce autophagy. In this study, we used an in vitro model of human monocyte derived macrophages (MDM) and primary murine alveolar macrophages (AM) that were infected with Mtb H37Rv and treated with rapamycin, loperamide, carbamazepine, verapamil and valproic acid 24h post-infection. We examined the induction of autophagy and the containment of Mtb within the autophagosomes by fluorescence microscopy, the gene expression of autophagy related proteins LC3 and ATG16L1 by real time PCR and the reduction in the intracellular Mtb burden by CFU. We found that rapamycin and valproic acid induced autophagy in 77.48 ± 13.29 and 88 ± 8.16 % of MDM, respectively, and in 70.62 ± 21.78 and 70 ± 6.83 % of AM, respectively, but did not improve Mtb growth control. Loperamide, carbamazepine and verapamil significantly induced autophagy in 70-90% of uninfected and infected human and murine macrophages and helped internalizing Mtb within autophagosomes; colocalization of Mtb with LC3 was larger than 60% in all treatments whereas in untreated cells it was of 34.73 ± 12.84 %. AM treated with carbamazepine, loperamide and verapamil decreased the intracellular bacillary burden up to 50% when administered in vitro. All drugs up-regulated the gene expression of LC3 and ATG16L1. To evaluate the capability of the drugs to pre-activate lung macrophages in vivo, we administered the three drugs intraperitoneally to uninfected mice, recovered the AM and infected them ex vivo. Loperamide treatment significantly reduced the intracellular growth of Mtb at Day 3 post infection without further treatment. Treatment with autophagy inducing-drugs has potential for use as adjunctive therapy in tuberculosis. Acknowledgements Funding: CONACYT-SALUD- 181008 Keywords: Autophagy, Macrophages, Tuberculosis, Loperamide, Carbamazepine Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015. Presentation Type: Poster Presentation Topic: Infectious and parasitic diseases Citation: Juárez E, Carranza C, Sánchez G, González M, Chávez J, Torres M and Sada E (2015). Autophagy inducing drugs contribute to control Mycobacterium tuberculosis infection. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00160 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 06 May 2015; Published Online: 14 Sep 2015. * Correspondence: Dr. Esmeralda Juárez, National Institute of Respiratory Diseases, Research in Microbiology, Mexico City, Federal District, 14080, Mexico, ejuarez@iner.gob.mx Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Esmeralda Juárez Claudia Carranza Guadalupe Sánchez Mitzi González Jaime Chávez Martha Torres Eduardo Sada Google Esmeralda Juárez Claudia Carranza Guadalupe Sánchez Mitzi González Jaime Chávez Martha Torres Eduardo Sada Google Scholar Esmeralda Juárez Claudia Carranza Guadalupe Sánchez Mitzi González Jaime Chávez Martha Torres Eduardo Sada PubMed Esmeralda Juárez Claudia Carranza Guadalupe Sánchez Mitzi González Jaime Chávez Martha Torres Eduardo Sada Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Iron acts as a second signal during coinfection with M. tuberculosis and N. brasiliensis leading to exacerbated M. tuberculosis disease outcome (MPF1P.769)

Abstract Previously, our laboratory has demonstrated that N. brasiliensis (Nb), an intestinal helminth, is able to exacerbate Mtb burden in coinfected Balb/c mice through generation of alternatively activated macrophages (AAMs). To examine the role of host genetic background in infection we performed coinfection experiments in C57Bl/6 mice. In contrast to Balb/c, coinfected C57Bl/6 mice were able to control Mtb burden comparable to mice infected with Mtb alone, despite mounting a Th2 response. This led us to hypothesize that a factor in addition to the generation of AAMs was responsible for the increased Mtb burden seen in the coinfection model. Concurrent experiments designed to understand Mtb survival within in vitro derived AAMs showed that IL-4 and IL-13 treatment was insufficient to cause differences in Mtb growth, compared to untreated macrophages. However the addition of iron significantly increased Mtb growth in AAMs. Iron, besides being important for Mtb growth; is also present in the lungs following tissue damage caused by Nb larval migration. To determine the role of iron during Nb infection we evaluated the expression of iron responsive genes in the murine lungs at 3, 5, and 7 days post infection. Balb/c and C57Bl/6 mice exhibited distinct profiles in response to Nb infection consistent with iron recycling and iron sequestration, respectively. Future studies aim to further understand the role of iron as important cofactor in regulating bacterial burden during coinfection.

Clonal Expansions of CD8+ T Cells with IL-10 Secreting Capacity Occur during Chronic Mycobacterium tuberculosis Infection

The exact role of CD8+ T cells during Mycobacterium tuberculosis (Mtb) infection has been heavily debated, yet it is generally accepted that CD8+ T cells contribute to protection against Mtb. In this study, however, we show that the Mtb-susceptible CBA/J mouse strain accumulates large numbers of CD8+ T cells in the lung as infection progresses, and that these cells display a dysfunctional and immunosuppressive phenotype (PD-1+, Tim-3+, CD122+). CD8+ T cell expansions from the lungs of Mtb-infected CBA/J mice were also capable of secreting the immunosuppressive cytokine interleukin-10 (IL-10), although in vivo CD8+ T cell depletion did not significantly alter Mtb burden. Further analysis revealed that pulmonary CD8+ T cells from Mtb-infected CBA/J mice were clonally expanded, preferentially expressing T cell receptor (TcR) Vβ chain 8 (8.2, 8.3) or Vβ 14. Although Vβ8+ CD8+ T cells were responsible for the majority of IL-10 production, in vivo depletion of Vβ8+ did not significantly change the outcome of Mtb infection, which we hypothesize was a consequence of their dual IL-10/IFN-γ secreting profiles. Our data demonstrate that IL-10-secreting CD8+ T cells can arise during chronic Mtb infection, although the significance of this T cell population in tuberculosis pathogenesis remains unclear.

Protection of mice from Mycobacterium tuberculosis by ID87/GLA-SE, a novel tuberculosis subunit vaccine candidate

Tuberculosis is a major health concern. Non-living tuberculosis (TB) vaccine candidates may not only be safer than the current vaccine (BCG) but could also be used to boost BCG to enhance or elongate protection. No subunit vaccines, however, are currently available for TB. To address this gap and to improve the global TB situation, we have generated a defined subunit vaccine by genetically fusing the genes of 3 potent protein Mtb antigens, Rv2875, Rv3478 and Rv1886, into a single product: ID87. When delivered with a TLR4 agonist-based adjuvant, GLA-SE, ID87 immunization reduced Mtb burden in the lungs of experimentally infected mice. The reduction in bacterial burden of ID87/GLA-SE immunized mice was accompanied by an early and significant leukocyte infiltration into the lungs during the infectious process. ID87/GLA-SE appears to be a promising new vaccine candidate that warrants further development.

New diagnostic methods for tuberculosis

During the last decade, laboratory tests for the detection of Mycobacterium tuberculosis (Mtb) have improved dramatically. Improvements in the ability to detect latent infection with Mtb, disease associated with Mtb, and strains resistant to commonly used antibiotics are reviewed. Advances in the detection of Mtb include light-emitting diode fluorescence microscopy, nucleic acid amplification of Mtb and drug-resistant strains, and more rapid liquid culture with adjunct drug susceptibility testing. In the detection of latent tuberculosis infection, interferon [gamma] release assays offer improved accuracy over the tuberculin skin test. The past 10 years have seen the most rapid growth in new diagnostics for Mtb in over a century. Although these tests offer improvements in the ability to detect Mtb, drug-resistant isolates, and those with latent tuberculosis infection, these improvements are counter-balanced by the need to deploy these tests in areas where Mtb burden is highest.

Pathogen-specific regulatory T cells impair protective immunity in tuberculosis (129.25)

Abstract Following aerosol infection with Mycobacterium tuberculosis (Mtb), Foxp3-expressing regulatory T cells (T regs) proliferate, accumulate at sites of infection, and suppress protective immunity. Here we use TCR transgenic CD4+ T cells specific for an immunodominant Mtb antigen (Ag85B240-254/I-Ab) to investigate the origin and specificity of T regs in tuberculosis. These cells robustly proliferated after transfer into Mtb-infected mice, but did not convert into Foxp3-expressing T regs. Instead, T regs expanded from the pool of Foxp3-expressing cells that existed prior to infection. To further examine the specificity requirements for responding T regs, we used a subset of these transgenic Mtb-specific T cells that co-expressed Foxp3. Only T regs that recognized Mtb antigen proliferated and accumulated at sites of infection and transfer of even small numbers of these cells into infected mice resulted in a higher Mtb burden in the lungs. Using Mtb-specific MHC class II tetramers to track the endogenous pathogen-specific T cell response, Mtb-specific T regs were found to mediate this effect by delaying the appearance of Mtb-specific, IFN-γ-producing CD4+ T cells in the lung, a delay that has been previously associated with impaired resistance to tuberculosis. Our results suggest that individuals containing increased numbers of pathogen-specific T regs will be more susceptible to severe disease.